Preface
Contents
Contributors
Part I: Molecular Biology and Bioinformatics Methods
Chapter 1: Generation of Markerless Gene Deletion Mutants in Listeria monocytogenes Using a Mutated pheS for Counterselection
1 Introduction
2 Materials
2.1 Gibson Assembly Design
2.2 Gibson Assembly Reaction
2.3 Transformation
2.4 Conjugation (Two-Parental Mating)
2.5 Plasmid Integration and Curing
2.6 Mutant Verification
3 Methods
3.1 Designation/Determination of the Insert Homologous Regions and Gibson Assembly Primer Design
3.2 Gibson Assembly Reaction
3.3 Transformation to a Donor E. coli Strain SM10
3.4 Conjugation (Two-Parental Mating)
3.5 Plasmid Integration (Pop-In)
3.6 Plasmid Curing (Pop-Out)
3.7 Mutant Verification
4 Notes
References
Chapter 2: A Rapid Fluorescence-Based Screen to Identify Regulators and Components of Interbacterial Competition Mechanisms in...
1 Introduction
1.1 Principle of Bacterial Competition Fluorescence (BaCoF)
1.2 Method Requirements
1.3 Applying BaCoF on a Vibrio parahaemolyticusT6SS
2 Materials
2.1 Media and Solutions
2.2 Strains and Plasmids
2.3 Equipment
3 Methods
3.1 Generating a Transposon Mutant Library
3.2 Calibrating BaCoF
3.3 Screening a Mutant Library
4 Notes
References
Chapter 3: Predicting Type III Effector Proteins Using the Effectidor Web Server
1 Introduction
2 Running Examples
2.1 Preparing the File of Genome ORFs
2.2 Preparing the File of Known Effectors (Note 1)
2.3 Running Effectidor
2.4 Analyzing Effectidor Predictions
3 Features Analysis (See Note 4)
4 Summary
5 Notes
References
Chapter 4: Assay for Type III Secretion in Escherichia coli
1 Introduction
2 Materials
2.1 Agar Plates
2.2 T3SS Assay
2.3 Sodium Dodecyl Sulfate (SDS) Polyacrylamide Gel
2.4 Immunoblotting
3 Methods
3.1 Growing Bacteria Under T3SS-Inducing Conditions
3.2 Separating the Secreted Fraction from the Bacterial Pellet
3.3 Electrophoresis and Coomassie Staining
3.4 Wet Transfer and Blotting
4 Notes
References
Chapter 5: Profiling of Secreted Type 3 Secretion System Substrates by Salmonella enterica
1 Introduction
2 Materials
2.1 Growth Media
3 Methods
4 Notes
References
Part II: Cell Culture and Organoid Models of Infection
Chapter 6: Analysis of SPI-1 Dependent Type III Secretion and Injection Using a NanoLuc Luciferase-Based Assay
1 Introduction
2 Materials
2.1 S. Typhimurium Culture
2.2 Plasmids for Expression of NLuc or HiBiT
2.3 Nano-Glo Luciferase Assay
2.4 Generation of an HeLa Cell Line Expressing LgBiT
2.5 Infection of HeLa-LgBiT Cells with S. Typhimurium
3 Methods
3.1 Salmonella SPI-1 Secretion Assay Using the Nano-Glo System
3.1.1 Salmonella Growth Conditions
3.1.2 Measurement of T3S Using Bacterial Supernatants
3.2 Salmonella Injection Assay Using the Nano-Glo System
3.2.1 Construction of a Mammalian Cell Line Expressing LgBiT
Initiate Lenti-X 293 T Cell Line Cultures from a Frozen Stock
Cultivation of HEK293T Cells
Transfection of the HEK293T Cells
Concentrating the Supernatant Using Lenti-X Concentrator
Determination of Lentiviral Titer
Transduction of HeLa Cells
Generation of Stable Transduced Cell Clones
Identification of LgBiT-Expressing HeLa Cell Clones
3.2.2 Salmonella Growth Conditions and Inoculum Preparation
3.2.3 Infection of HeLa-LgBiT Cells and Measurement of Injection Kinetics
3.2.4 Infection of HeLa-LgBiT Cells and Measurements of Injection (End-Point Measurement)
4 Notes
References
Chapter 7: Mycobacterium tuberculosis Infection of THP-1 Cells: A Model for High Content Analysis of Intracellular Growth and ...
1 Introduction
2 Materials
2.1 THP-1 Cell Culture
2.2 Mtb Culture
2.3 Infection
2.4 Fixation and Staining
2.5 Analytical Programs and Software
3 Methods
3.1 HCS of Antituberculosis Candidates on Intracellular Mtb
3.1.1 Cell Culture
3.1.2 Batch Infection
3.1.3 Two-Step Infection
3.1.4 Fixation and Nuclear Staining
3.1.5 HCS Platform Setup and Scanning
3.1.6 HCS Data Analysis
3.1.7 Zβ² Factor Analysis
3.2 HCS of Mtb Mutant Libraries Using DMN-Tre
3.2.1 Batch Infection
3.2.2 Two-Step Infection
3.2.3 Fixation and DAPI Staining
4 Notes
References
Chapter 8: Bone Marrow-Derived Macrophage (BMDM) Infection by Listeria monocytogenes
1 Introduction
2 Materials
2.1 Preparation of L929-Conditioned Medium
2.2 Preparation of BMDMs
2.3 Infection of BMDMs by L. Monocytogenes
2.4 Intracellular Growth Analysis of L. monocytogenes in BMDMs
3 Methods
3.1 Preparation of L929-Conditioned Medium
3.2 Preparation of BMDMs
3.3 Infection of BMDMs by Listeria monocytogenes
3.4 Intracellular Growth Analysis of L. monocytogenes in BMDMs
4 Notes
References
Chapter 9: Preparation and Inflammasome Activation of Murine Bone Marrow-Derived and Resident Peritoneal Macrophages
1 Introduction
2 Materials
2.1 Mice
2.2 Reagents
2.3 Materials
3 Methods
3.1 Preparation of BMMs
3.2 Preparation of rPMs
3.3 Evaluation of Purity of Macrophages
3.4 Stimulation with Inflammasome Activators
4 Notes
References
Chapter 10: Flow Cytometry-Based Single Cell Analyses of Bacterial Adaptation to Intracellular Environments
1 Introduction
1.1 Flow Cytometry for Single Cell Infection Biology
2 Materials
2.1 Bacterial Strains, Host Cells, Bacterial Infection
2.2 Instrument and Filter Sets
2.3 Software
3 Methods
3.1 Protocol for Preparing and Measuring Bacteria In Vitro
3.2 Protocol for Preparing and Measuring Bacteria Liberated from Host Cells (HeLa, RAW264.7, U937, etc.)
3.3 Protocol for Preparing and Measuring Infected Host Cells (HeLa, RAW264.7, U937, etc.)
4 Notes
References
Chapter 11: Quantification of Microbial Fluorescent Sensors During Live Intracellular Infections
1 Introduction
2 Materials
2.1 Bone Marrow-Derived macrophages (BMDMs)
2.2 Bacteria Encoding a Fluorescent Sensor
2.3 BMDM Infections and Microscopy
2.4 Image Analysis
3 Methods
3.1 Preparation of Bone Marrow-Derived Macrophages
3.2 Preparation of Fluorescent Sensor-Expressing Bacteria
3.3 Infection of Bone Marrow-Derived Macrophages
3.4 Microscopy of Bone Marrow-Derived Macrophages
3.5 Analysis of Microscopy Images (Fig. 1)
3.6 Microscopy Optimization for Multiple Reporters
4 Notes
References
Chapter 12: Dissecting Human Blood Immune Cells Response to Intracellular Infection Using Single-Cell RNA Sequencing
1 Introduction
1.1 Abbreviations
2 Materials
2.1 PBMC Isolation from Human Blood Sample
2.2 Monocyte Enrichment
2.3 Bacterial Growth
2.4 Bacterial Preparation, PBMC Infection, and Preparation for scRNA-Seq
3 Methods
3.1 PBMC Isolation from Human Blood Sample
3.1.1 Starting from Human Whole Blood Sample
3.1.2 Starting from Human Leukocyte Enriched Fraction
3.1.3 Lymphoprep Extraction
3.2 Monocyte Enrichment
3.3 Bacteria Growth
3.4 PBMC/Monocyte Infection
3.4.1 Bacterial Preparation
3.4.2 PBMC/Monocyte Infection
PBMC Infection
Adaptations for Monocytes Infection
3.5 Cell Preparation for scRNA-Seq
3.5.1 For PBMCs
3.5.2 Suggested Adaptations for Monocytes Only
3.6 Bacterial Culture Verification
4 Notes
References
Chapter 13: Salmonella enterica Infection of Human and Mouse Colon Organoid-Derived Monolayers
1 Introduction
2 Materials
2.1 Collection of L-WRN-Conditioned Medium
2.2 Isolation of Colon Crypts from Mouse Tissue
2.3 Isolation of Colon Crypts from Human Tissue
2.4 Passaging Colon Organoids
2.5 Generating 2D Colon Epithelial Cell Monolayers
2.6 S. enterica Infection of 2D Colon Epithelial Cell Monolayers
2.7 Quantifying S. enterica Adherence and Invasion of 2D Colonoids Using the Gentamicin Protection Assay
3 Methods
3.1 Collection of L-WRN-Conditioned Medium
3.2 Isolation of Colon Crypts from Mouse Tissue
3.3 Isolation of Colon Crypts from Human Tissue
3.4 Passaging Colon Organoids
3.5 Generating 2D Colon Epithelial Cell Monolayers
3.6 S. enterica Infection of 2D Colon Epithelial Cell Monolayers
3.7 Quantifying S. enterica Adherence and Invasion of 2D Colonoids Using the Gentamicin Protection Assay
4 Notes
References
Part III: In Vivo Models of Infection
Chapter 14: Analysis of Salmonella enterica Adhesion to Leaves of Corn Salad or Lettuce
1 Introduction
1.1 Gastrointestinal Infections by Contaminated Vegetables
1.2 Bacterial Adhesiomes
2 Materials
2.1 Sterilization of Salad Seeds
2.2 Cultivation of Salad
2.3 Adhesion Assay
3 Methods
3.1 Sterilization of Corn Salad Seeds
3.2 Sterilization of Lettuce Seeds
3.3 Cultivation of Salad Species
3.4 Bacterial Culture for Adhesion Assay
3.5 Adhesion Assay
4 Analysis
5 Notes
References
Chapter 15: Methods for Using the Galleria mellonella Invertebrate Model to Probe Enterococcus faecalis Pathogenicity
1 Introduction
2 Materials
2.1 Reagents and Equipment Required
2.2 G. Mellonella Last Instar Larvae (Approximately 6 Weeks Old)
2.3 Inoculum Containing Strain of Interest
3 Methods
3.1 To Test for Bacterium Virulence Determinants
3.2 To Test Antimicrobials Efficacy Against Bacterial Infection
4 Notes
References
Chapter 16: Murine Soft Tissue Infection Model to Study Group A Streptococcus (GAS) Pathogenesis in Necrotizing Fasciitis
1 Introduction
1.1 Group A Streptococcus
1.2 Animal Models for the Study of Infectious Diseases-General Considerations
1.3 Animal Models for Studying GAS Diseases
1.4 Human GAS NF
1.5 A Murine Model for Studying GAS NF Pathogenesis
2 Materials
2.1 GAS Strains and Bacterial Culturing and Preparation for Infection of Mice
2.2 Infection of Mice
2.3 Quantification of GAS CFU in Skin and Spleen of Infected Mice
2.4 Sample Processing for Histological Analysis
2.5 Immunofluorescence Staining
2.6 Hematoxylin and Eosin Staining
3 Methods
3.1 Culturing and Preparation of Bacteria for Infection of Mice
3.2 Determination of Mice Survival Using a Mouse Model of Lethal Human Soft-Tissue Infection
3.3 A Sublethal Mouse Model of Human Soft-Tissue Infection for CFU Determination and Histological Analysis
3.4 Quantification of GAS CFU in Skin and Spleen of Infected Mice
3.5 Sample Processing for Histological Analysis
3.6 Immunofluorescence Staining of Frozen Tissue Sections
3.7 Hematoxylin and Eosin Staining
4 Notes
References
Chapter 17: Mouse Model to Study Salmonella-Induced Colitis
1 Introduction
1.1 Salmonella Induced Colitis
1.2 Host Resistance to S. Typhimurium Infection
1.3 Microbiota and Antibiotic Treatment
2 Materials
2.1 Mice
2.2 Salmonella entericaserovar Typhimurium (or Other NTS Serovars)
2.3 Consumables
2.4 Chemicals and Media
2.5 Equipment
3 Methods
3.1 Streptomycin Treatment
3.2 Inoculum
3.3 Oral Infection of Mice
3.4 Monitoring Fitness of Mice
3.5 Collection of Fecal Pellets and Processing During the Time Course of Infection
3.5.1 Collection of Fresh Fecal Pellets
3.5.2 Quantification of the Bacterial Load
3.5.3 Quantification of Inflammation Marker Lcn2
3.6 Collecting Tissues at the End of the Experiment
3.7 Sample Processing for Quantification of the Bacterial Organ Load
3.8 Histopathology Scoring
4 Notes
References
Chapter 18: Analysis of Salmonella Typhi Pathogenesis in a Humanized Mouse Model
1 Introduction
2 Materials
2.1 Humanized Mice-Sources
2.2 Source of S. Typhi
2.2.1 Characterized Clinical Isolates
2.2.2 Presence of Vi Antigen
2.2.3 Multidrug Resistance Status
2.3 Growth of S. Typhi
2.4 Infection of CD34+ HU-NSG and NSG Mice
2.5 Organ Harvest and Blood Collection
2.6 Transposon Library Construction
3 Methods
3.1 Biosafety and Animal Husbandry
3.1.1 Humanized Mice
3.1.2 Salmonella enterica
3.1.3 Laboratory Animal Husbandry Staff
3.2 Vi Agglutination Assay
3.3 Determination of S. Typhi Inoculum for CD34+ Hu-NSG Infection
3.3.1 Determination of S. Typhi Inoculum for Competitive Infections
3.4 Infection of CD34+ Hu-NSG Mice or NSG Mice
3.5 Blood Collection
3.6 Organ Homogenization for CFU Determination
3.7 Organ Preservation
3.8 Genetic Manipulation of Salmonella enterica
3.9 Transposon Library Construction, Infection, and Analysis
3.9.1 S. Typhi Transposon Library Construction
3.9.2 Recovery and Archiving of Input´´ andOutput´´ Samples
3.9.3 Isolation of DNA for TraDIS
3.9.4 DNA Shearing
3.9.5 End Repair
3.9.6 C-Tailing
3.9.7 PCR1
3.9.8 PCR2
3.9.9 PCR for Sequencing
3.9.10 SPRI Size Selection
3.9.11 Quantification
3.9.12 Next-Generation Sequencing
4 Notes
References
Chapter 19: In Vivo Tracking of Bacterial Colonization in Different Murine Models Using Bioluminescence: The Example of Salmon...
1 Introduction
2 Materials
2.1 Tn7 Chromosomal Integration of Reporter Fusions
2.2 Inoculum Preparation
2.3 Mouse Models
2.4 In Vivo Experiment and Bacterial Numeration
3 Methods
3.1 Construction of a No Promoter (NoP) Reporter Transcriptional Fusion (See Note 4)
3.2 Chromosomal Integration of Reporter Fusions (See Note 5)
3.3 Preparation of Inocula (See Note 7)
3.4 Preparation of Animals
3.5 Mice Infection, Anesthesia, and Imaging
3.6 Bioluminescence Quantification
4 Notes
References
Chapter 20: Two In Vivo Models to Study Salmonella Asymptomatic Carrier State in Chicks
1 Introduction
2 Materials
2.1 Bacterial Strain
2.2 Inoculum Preparation
2.3 Animals
2.4 In Vivo Experiment
2.4.1 Feed
2.4.2 Battery Cages
2.4.3 Cage
2.4.4 Large Isolator
2.4.5 Inoculation
2.4.6 Euthanasia
2.4.7 Autopsy
2.5 Bacterial Numeration
3 Methods
3.1 Preparation of a Frozen Inoculum
3.2 Animal Handling Before Inoculation
3.3 Checking for the Absence of Salmonella in Chicks
3.4 Inoculation
3.5 Sample Recovery from Live Animals
3.5.1 Fresh Fecal Samples
3.5.2 Blood Samples
3.6 Post-mortem Sample Recovery
3.6.1 Euthanasia
3.6.2 Beginning of the Necropsy
3.6.3 Blood Sampling
3.6.4 Recovery of Internal Organs
3.6.5 Cecal Tissue and Cecal Content Recovery
3.7 Bacterial Numeration
4 Notes
References
Index